Trak1 is a novel regulator of mitochondrial fusion and cellular homeostasis Open Access

Lee, Crystal (2015)

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Trafficking protein, kinesin-binding 1 (Trak1) is a ubiquitously expressed protein functioning in two separate cellular pathways, mitochondrial transport and endosome-to-lysosome trafficking. Trak1 mutations are linked to several disease states. In mice, a homozygous frameshift mutation in the Trak1 gene causes a recessively transmitted form of hypertonia. In humans, elevated levels of Trak1 protein expression have been reported in several types of cancers and a Trak1 variant is linked to childhood absence epilepsy. However, the pathogenic mechanisms of Trak1 in these diseases states remain unknown. In my dissertation work, I identified a new function for Trak1 as a regulator of mitochondrial fusion independent of its role in mitochondrial transport and I found Trak1 to be essential to mitochondrial health and cell survival under cellular stress conditions. Furthermore, I found that the hypertonia-associated mutation of Trak1 exhibits partially disrupted targeting to mitochondria, suggesting a novel mechanism of hypertonia pathogenesis. In addition, I show that the Drosophila melanogaster orthologue of Trak1, Milton, is essential for viability and identified a functional role for Milton in the endocytic pathway, establishing the conserved dual functions of Trak1 and Milton. Together the work described in this dissertation, reveals a novel function for Trak1 in mitochondrial fusion and cellular homeostasis, and demonstrates that Trak1 is critical to both cellular and organismal health.

Table of Contents

Chapter 1. Introduction and background

1.1. Opening remarks. 2

1.2. Trak1 protein. 2

1.2.1. Trak1 is part of the HAPN domain family of proteins that also includes

GRIF-1 and HAP1. 7

1.2.2. Milton, the Drosophila homolog of Trak1. 12

1.3. Endocytic trafficking and regulation. 14

1.3.1. Consequences of endosomal-lysosomal pathway dysfunction. 18

1.4. Mitochondria. 21

1.4.1. Structure of mitochondria. 22

1.4.2. Functions of the mitochondria. 24

1.4.3. Mitochondrial dynamics: fusion, fission, transport, and mitophagy. 28

1.4.4. Mitochondrial fusion. 29

1.4.5. Mitochondrial fission. 32

1.4.6. Mitophagy. 33

1.4.7. Mitochondrial transport and regulation. 35

1.4.8. Consequences of mitochondrial dysfunction.. 41

1.5. Trak1 mutations are linked to disease states. 44

1.5.1. Hypertonia. 45

1.5.2. Childhood absence epilepsy. 48

1.5.3. Gastric and colorectal cancer. 50

1.6. Hypotheses and organizational overview. 51

Chapter 2. Hypertonia-linked protein Trak1 is a novel regulator of mitochondrial fusion

2.1. Abstract. 64

2.2. Introduction. 65

2.3. Results. 67

2.3.1. Deletion of Trak1 results in mitochondrial fragmentation. 67

2.3.2. Trak1 functions in regulation of mitochondrial fusion. 68

2.3.3. Hypertonia-linked mutation impairs Trak1 mitochondrial localization and function. 69

2.3.4. Trak1 overexpression induces mitochondrial hyperfusion. 70

2.3.5. Trak1 functions upstream of Mitofusins in regulating mitochondrial fusion. 72

2.3.6. Trak1 overexpression promotes mitochondria tethering in Mitofusin-deficient cells. 74

2.3.7. Trak1 is essential for stress-induced mitochondrial hyperfusion and pro-survival response. 75

2.4. Discussion. 75

2.5. Materials and methods. 79

2.5.1. Cell culture, Transfection, and Immunoblotting analysis. 79

2.5.2. Plasmids and Antibodies. 79

2.5.3. Immunofluorescence confocal microscopy. 80

2.5.4. MitoDendra2 assay and Analysis of fusion. 81

2.5.5. 3D-Structured illumination microscopy (SIM). 81

2.5.6. Electron microscopy. 82

2.5.7. Subcellular fractionation. 82

2.5.8. Stress-induced mitochondrial hyperfusion and apoptosis detection. 83

2.5.9. Analysis of mitochondrial morphology. 83

2.5.10. Statistical analysis. 83

2.6. Acknowledgements. 84

Chapter 3. Characterizing the role of Milton in Drosophila development

3.1. Abstract. 110

3.2. Introduction. 110

3.3. Results. 113

3.3.1. Milton null flies display early larval lethality. 113

3.3.2. Milton is expressed in all stages of Drosophila development. 114

3.3.3. Milton mutants do not affect pigment granule trafficking or photoreceptor degeneration. 115

3.3.4. Milton is involved in signaling pathways in the Drosophila wing. 116

3.3.5. Milton mutants have reduced lifespan and locomotor activity. 118

3.4. Discussion. 118

3.5. Materials and methods. 120

3.5.1. Fly strains. 120

3.5.2. RT-PCR. 121

3.5.3. Lifespan assay. 121

3.5.4. Locomotor assay. 121

Chapter 4. Summary of findings and future directions

4.1. Summary of findings. 133

4.2. Future directions. 136

4.2.1. Does loss of Trak1 affect the function of mitochondria?. 136

4.2.2. How does Trak1 regulate two separate cellular pathways?. 138

4.2.3. What is the specific mechanism of Trak1 mediated mitochondrial tethering?. 140

4.2.4. Can Trak1 be used therapeutically, such as to rescue the excessive mitochondrial fragmentation and neuronal dysfunction seen in neurodegenerative diseases?. 141

4.2.5. Does Trak1 regulate other aspects of mitochondrial dynamics such as fission or mitophagy?. 142

4.2.6. What is the pathogenic mechanisms of Trak1 hyrt associated hypertonia?. 144

4.2.7. Does Milton share functional homology with Trak1 in flies?. 146

4.3. Final words. 147

References. 150

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